The analysis of protein deficiency in patients hospitalized due to

The analysis of protein deficiency in patients hospitalized due to

Postępy Nauk Medycznych, t. XXVII, nr 11, 2014
©Borgis
*Błażej Rozpłochowski1, 2, Olga Słyńko-Medoń1, 2, Michał Chojnicki1, 3, Agnieszka Seraszek-Jaros4,
Arleta Kowala-Piaskowska1, 2, Iwona Mozer-Lisewska1, 2
The analysis of protein deficiency in patients hospitalized due
to Clostridium difficile infection
Analiza deficytów białkowych u osób hospitalizowanych z powodu biegunki
związanej z Clostridium difficile
1
Department of Infectious Diseases, J. Struś Multispecialist City Hospital, Poznań
Head of Department: prof. Iwona Mozer-Lisewska, MD, PhD
2
Department and Clinic of Infectious Diseases, Poznań University of Medical Sciences
Head of Department: prof. Iwona Mozer-Lisewska, MD, PhD
3
Department of Biology and Environmental Protection, Poznań University of Medical Sciences
Head of Department: prof. Krzysztof Wiktorowicz, PhD
4
Department of Bioinformatics and Computational Biology, Chair of Clinical Pathomorphology,
Poznań University of Medical Sciences
Head of Department: prof. Elżbieta Kaczmarek, PhD
Key words
Summary
diarrhoea, Clostridium difficile, protein,
albumins
Introduction. Clostridium difficile infections are one of the major causes of bacterial
diarrhoea, particularly in patients aged over 65 years. Hospitalization time depends not
only on the severity of the diarrhoea, but mainly on the decompensation of the general
status of patients with concurrent chronic diseases. Protein-losing enteropathy, a complication resulting from the exposure of the intestines to toxins released by C. difficile, leads
to a considerable loss of protein, particularly the albumin fraction, and deterioration of the
patient’s general health.
Aim. The aim of this study was to evaluate protein deficiency in patients with CDI and to
identify the risk factors for such disorders related to demographic and iatrogenic parameters.
Material and methods. The retrospective analysis of C. difficile-associated diarrhoea (CDAD) among patients hospitalized at the Department of Infectious Diseases
(J. Struś Multispecialist City Hospital, Poznań) in 2013.
An episode of CDI was diagnosed based on the recommendations of the experts.
The aetiology of Clostridium difficile-associated diarrhoea was confirmed using
TECHLAB immunoenzymatic tests, i.e. a rapid membrane-based assay detecting the glutamate dehydrogenase (GDH) antigen and a rapid assay detecting toxins A and B from
C. difficile. Protein metabolism was evaluated based on the measured total protein or
albumin levels in blood plasma.
Protein disorders were analysed depending on the age and sex of patients, the duration and
severity of the diarrhoea, and inflammatory markers, i.e. levels of C-reactive protein and leukocytosis. The correlation between the creatinine levels and risk factors for CDI was also tested.
Test results were analysed using the Shapiro-Wilk and Mann-Whitney tests.
Results. Deficiency in total protein and albumin levels was found in 28 (71,8%) of patients from the studied population. Total protein deficiency correlated with deficiency of
the albumin fraction, while the severity of the deficiency depended on the duration of the
diarrhoea, and not on the daily number of passed stools. No evidence supporting the assumed correlation between the protein deficiency and the patient’s age, renal function or
the levels of inflammatory markers (C-reactive protein and leukocytosis) was found.
Conclusions. CDI is a systemic disease correlated with protein loss. Because of the
patient’s age and numerous concomitant diseases, CDI poses a serious threat to the patient’s life, and in each case requires the evaluation of protein metabolism.
Słowa kluczowe
biegunka, Clostridium difficile, białko,
albumina
Address/adres:
*Błażej Rozpłochowski
Department and Clinic of Infectious Diseases
University of Medical Sciences
ul. Szwajcarska 3, 61-285 Poznań
tel. +48 (61) 873-93-76
fax +48 (61) 873-92-90
[email protected]
Streszczenie
Wstęp. Zakażenia Clostridium difficile są jedną z dominujących przyczyn biegunki bakteryjnej, zwłaszcza wśród osób po 65. roku życia. Czas trwania hospitalizacji zależy nie tylko
od nasilenia biegunki, ale zwłaszcza od dekompensacji stanu ogólnego osób z towarzyszącymi chorobami przewlekłymi. Z powodu enteropatii wysiękowej, będącej powikłaniem
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Błażej Rozpłochowski et al.
ekspozycji jelit na toksyny C. difficile, dochodzi do znacznego ubytku białka, a zwłaszcza
frakcji albumin, co wiąże się z pogorszeniem stanu ogólnego chorujących osób.
Cel pracy. Celem badania była ocena deficytów białkowych u pacjentów leczonych
z powodu biegunki związanej z Clostridium difficile oraz wyznaczenie czynników ryzyka
takich zaburzeń wśród parametrów demograficznych oraz jatrogennych.
Materiał i metody. Retrospektywna analiza zachorowań na biegunkę związaną
z C. difficile CDI wśród pacjentów Oddziału Zakaźnego (Wielospecjalistyczny Szpital Miejski im. J. Strusia, Poznań) hospitalizowanych w 2013 roku.
Epizod CDI rozpoznawano w oparciu o rekomendacje ekspertów. Etiologię biegunki
Clostridium difficile potwierdzono testami immunoenzymatycznymi TECHLAB, tj. szybkim
testem membranowym na obecność antygenu dehydrogenazy glutaminowej (GDH) oraz
szybkim testem do detekcji toksyny A i B C. difficile.
Ocenę gospodarki białkowej prowadzono w oparciu o oznaczenie stężenia białka całkowitego lub albumin w surowicy krwi.
Zaburzenia białkowe analizowane były w zależności od wieku i płci pacjentów, czasu
trwania i nasilenia biegunki oraz wykładników stanu zapalnego takich jak stężenia białka
C-reaktywnego oraz leukocytozy. Sprawdzono również korelację pomiędzy stężeniem
kreatyniny oraz uznawanych za czynnik ryzyka wystąpienia CDI.
Zgodność z rozkładem normalnym sprawdzono za pomocą testu Shapir-Wilka. Porównanie miedzy grupami kobiet i mężczyzn wykonano za pomocą testu Manna-Whitney’a.
Wyniki. W badanej populacji potwierdzone niedobory stężenia białka całkowitego oraz
albumin wystąpiły u 28 (71,8%) chorych. Niedobór białka całkowitego korelował z niedoborem frakcji albumin, zaś głębokość niedoboru była zależna nie od dobowej liczby
stolców, ale od czasu trwania biegunki. Nie potwierdzono również przypuszczenia, że niedobór białka jest zależny od wieku pacjenta, czynności nerek lub wysokości wskaźników
stanu zapalnego (białka C-reaktywnego oraz leukocytozy).
Wnioski. Biegunka związana z C. difficile jest chorobą ogólnoustrojową, która łączy się
z utratą białka. Z uwagi na wiek pacjentów oraz liczne choroby współistniejące, biegunka
związana z C. difficile jest poważnym zagrożeniem dla życia pacjentów i w każdym przypadku wymaga diagnostyki gospodarki białkowej.
INTRODUCTION
Clostridium difficile is a Gram-positive anaerobic,
toxigenic and spore-forming bacterium. It was described for the first time in the 1940s as a nonpathogenic component of the intestinal microbial flora in
healthy new-born infants (1). After broad-spectrum antibiotics were introduced in medical practice, attention
was drawn to the pathogenic nature of this bacterium
due to the growing number of patients suffering from
pseudomembranous colitis (2). Currently, it is known
that the spectrum of clinical manifestations for Clostridium difficile includes asymptomatic colonization, but
also cases of colitis of various severity, including watery diarrhoea, pseudomembranous inflammation and
toxic megacolon (2). The clinical picture is largely determined by the immune system function in individual
patients, but disorders in the composition of intestinal
microbial flora are the key factor for the development
of Clostridium difficile infections (CDI) (3). For this reason, the inducing factors, e.g. the use of antibiotics,
proton pump inhibitors, immunosuppressants, as well
as hospitalizations or concomitant diseases are the
major risk factors for this disease (3).
The worldwide incidence of CDI has been increasing
for several decades, particularly in the developed countries (4). This process is attributed to the increased incidence of the above-mentioned risk factors, but also to
the emergence of hypervirulent strains, and the ageing
of the population (3, 4).
In 2013 in Poland 4716 cases of the disease were
reported, which makes Clostridium difficile second to
Salmonella spp. as the most frequent bacterial cause
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of intestinal infections. The prevalence of CDI is 12.24
per 100 000 people, and is currently lower than in other
developed countries (5).
The rate of increase and scale of the problems associated with Clostridium difficile stimulate the need
for the development and updating of guidelines on
the classification, diagnostics, treatment and prevention of CDI. Despite the growing number of community-acquired CDI cases (CA-CDI), the disease is usually contracted through contact with health care and is
one of the major iatrogenic types of infection (Health
Care-Associated Infection – HCAI) (4). According to reports from the United States, including the analysis of
the 5 most frequent HCAIs, these diseases generate
annual costs of USD 9.8 bn, of which CDI is responsible for 15.4% (6).
In everyday medical practice, protein disorders
seem to be frequent among patients hospitalized due
to CDI. Proteins are involved in all processes at the
cellular level and the entire body. Proteins take part
in the coordination of life functions at multiple levels:
the generation and transmission of information, buffering changes in the levels of other biologically active
substances, the control of growth and differentiation,
and cell-cell interactions. Albumins are mainly responsible for the maintenance of the oncotic pressure of
plasma and its correct volume. They are also the carrier proteins transporting free fatty acids, bilirubin, certain drugs, metal ions and hormones. Albumins also
form the protein reserve in the body and are used as
a source of amino acids for the synthesis of proteins
in cells of organs other than the liver (7). For this rea-
The analysis of protein deficiency in patients hospitalized due to Clostridium difficile infection
son protein deficiency frequently results in peripheral
oedema, ascites and hypotension. The mechanisms
responsible for protein deficiency in patients with CDI
and caused by protein-losing enteropathy were described more than twenty years ago (8). However, the
results of studies analysing the incidence of protein
disorders among patients with CDI were not conclusive
and indicated the need for further research (9). Currently available reports contain the analysis of hypoproteinaemia in the context of evaluation of the severity of CDI recurrence (9). We have retrieved only one
report evaluating protein deficiency in patients treated
for CDI (10). Therefore, our study may set new trends
in research and contribute to updating currently available guidelines.
AIM
ity and range of the test was 0-220 mg/l. The range for
biological reference values was 0-10 mg/l.
Test results were analysed using the Shapiro-Wilk
and Mann-Whitney tests.
Protein disorders were analysed depending on the
age and sex of patients, the duration and severity of
the diarrhoea, and inflammatory markers, i.e. levels of
C-reactive protein and leukocytosis. We also tested the
correlation between the creatinine levels and risk factors for CDI.
RESULTS
CDI was diagnosed in 77 hospitalized patients. Protein
metabolism was evaluated in 39 patients from the study
group. Women were in the majority 22 vs. 17 (56 vs. 44%).
The mean age of patients was 73.21 years (range
18-99 years, standard deviation 17.8 years) (fig. 1).
The aim of this study was to evaluate protein deficiency in patients with CDI and to identify the risk factors for such disorders related to demographic and iatrogenic parameters.
MATERIAL AND METHODS
We carried out a retrospective analysis of CDI
among patients hospitalized at the Department of Infectious Diseases in 2013.
An episode of CDI was diagnosed based on the recommendations of the Polish National Programme of
Antibiotic Protection, released in 2011, which are in line
with the guidelines of the European Society for Clinical Microbiology and Infectious Diseases. According
to the guidelines, the diagnosis of CDI was based on
bacterial culture to confirm the presence of C. difficile
in faeces, or detecting bacterial antigens or metabolites (11, 12).
The etiology of CDI was confirmed using TECHLAB
immunoenzymatic tests, i.e. a rapid membrane-based
assay detecting the glutamate dehydrogenase (GDH)
antigen (sensitivity: 92.8%, specificity: 92.6%, positive predictive value: 78.6%, negative predictive value: 98.7%, correlation: 92.6%) and a rapid assay detecting toxins A and B from C. difficile (sensitivity: 90.2%,
specificity: 99.7%, positive predictive value: 98.6%,
negative predictive value: 97.9%, correlation: 98.0%).
Protein metabolism was evaluated based on the
measured total protein and/or albumin levels in blood
plasma.
Total protein levels were determined using a colorimetric method (burette reaction) by measuring the increase in absorbance at wavelength 540 nm. The sensitivity and range of the test was 1.0-15.0 mg/dl. The range
for biological reference values was 6.2-8.5 g/dl.
Albumin levels were determined using a colorimetric
method by measuring the increase in colour intensity
at the wavelength of 630 nm. The sensitivity and range
of the test was 0.5-8.0 g/dl. The range for biological
reference values was 3.5-5.3 g/dl.
CRP levels were measured using the latex particle
enhanced immunoturbidimetric method. The sensitiv-
Fig. 1. Distribution of the study group (in per cent) by age.
Mean duration of diarrhoea was 9.24 days. The mean
number of stools reported per day was 6.61 (max. 19).
For most of the patients this was the first episode of CDI,
but the maximum number of recurrences was 3 (fig. 2).
Diarrhoea was associated with increased levels of
inflammatory markers. The mean level of C-reactive
protein was 102 mg/dl, and the maximum CRP level
was above 240 mg/dl. Importantly, the increase in
C-reactive protein level was not associated with the increased leukocyte count (mean 14.57 G/L), and only in
one case was it 36.20 G/L (data not shown).
Fig. 2. Distribution of the study group (in per cent) by the number of
CDI episodes.
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Błażej Rozpłochowski et al.
Decreased levels of total protein or the albumin
fraction were found in 28 (71.8%) patients. Mean total
protein level was 5.32 g/dl, and 2.85 g/dl for albumins.
In nine (28,125%) patients the albumin level was below
2.5 g/dl (fig. 3).
Table 1. Correlation between protein loss and/or albumin loss
and the age of the patient, duration of the diarrhoea, number of
stools, eGFR, leukocyte count, C-reactive protein level, hematocrit level and creatinine level.
N
Correlation
coefficient r
% protein loss & age
28
-0.1470
0.4553
% protein loss & duration of diarrhoea
23
0.0055
0.9801
Analysed parameters
p
% protein loss & creatinine
28
0.1425
0.4695
% protein loss & eGFR
28
-0.1574
0.4238
% protein loss & number of stools
27
0.1029
0.6096
% protein loss & leukocytes
28
0.0115
0.9537
% protein loss & CRP
28
-0.0761
0.7004
% protein loss & HCT
28
0.0454
0.8184
% albumin loss & age
29
0.1476
0.4447
% albumin loss & duration
of diarrhoea
24
0.6567
0.0281
% albumin loss & creatinine
29
0.0489
0.8010
% albumin loss & eGFR
29
-0.1097
0.5712
Fig. 3. Distribution of the study group (in per cent) depending on the
albumin plasma level.
% albumin loss & number of stools
29
0.0612
0.7526
% albumin loss & leukocytes
29
0.2529
0.1856
The correlation between the duration of diarrhoea
and albumin deficiency (p < 0.05) was confirmed in
the study group (fig. 4) (tab. 1).
The analysis of total protein and the albumin fraction
levels confirmed that the protein loss mainly concerns the
albumin fraction, and there is a strong correlation between
the loss of total protein and albumins (p = 0.0281).
No correlation was found between the number of
stools, inflammatory markers (leukocytosis, CRP levels), GFR, hematocrit and age, sex or loss of total protein or albumin (tab. 1).
The correlation between the use of individual antibiotics and the incidence of CDI is presented in table 2.
According to the table, the most frequently used antibiotics were ceftriaxone, amoxicillin, ciprofloxacin and
cefuroxime. Interestingly, one hospitalized patient had
an episode of CDI following the use of metronidazole.
% albumin loss & CRP
28
0.0151
0.9393
% albumin loss & HCT
29
0.0628
0.7463
DISCUSSION
CDI is a growing epidemiological problem in Poland
and other countries (5, 13). In 2013 the prevalence
of CDI in Poland was estimated at 12.24 cases per
100 000 people. Because the obligation to report intes-
Fig. 4. Correlation between albumin loss and duration of diarrhoea.
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Table 2. Correlation between the use of individual antibiotics
and incidence of CDI.
Antibiotic taken
Number
of patients
% of patients
Ceftriaxone
4
18.18%
Ciprofloxacinum
3
13.63%
Amoxicillinum, Acidum clavulanicum
3
13.63%
Sulfamethoxazolum, Trimethoprimum
2
9.09%
Metronidazolum
1
4.55%
Piperacillin, Tazobactam
1
4.55%
Cefuroximum
1
4.55%
Not specified
7
31.82%
tinal infections caused by Clostridium difficile in Poland
was introduced in early 2013, it is impossible to describe the epidemiological trend in an objective fashion (5). However, epidemiological data from hospital
departments indicate a clear and significant increase
in the number of CDI cases. For this reason Clostridium
difficile is regarded as an alert pathogen. For example,
The analysis of protein deficiency in patients hospitalized due to Clostridium difficile infection
among patients discharged from hospitals in the United
States of America, the number of diagnosed cases of
CDI increased from 3.82/1000 in 2000 to 8.75/1000 in
2008 (13). A significant increase was reported among
patients aged over 65 years (13).
Results from our study also indicated a significantly
higher prevalence in this age group. Mean patient age
was 72.21 years, and patients aged over 65 years accounted for more than 75% of the studied population.
This result correlates with findings by other researchers,
who estimate that the risk of infection in people older than
65 years is 20-times greater than in people under 20 years
of age (14). For this reason being over 70 years of age is
one of the risk factors for a severe course of CDI (9).
In 1996 Dansinger et al. studied the differences between asymptomatic colonization and diarrhoea associated with the presence of toxins A and B from C. difficile,
and found a significant protein loss leading to malnutrition in patients with diarrhoea, but no protein loss in
asymptomatic patients (8). The severity of malnutrition
was not correlated with age, but resulting complications
were correlated with concomitant diseases (8).
Data suggest that when hospitalizing elderly patients
with CDI particular attention should be paid to concomitant diseases and disorders resulting from them, as
well as to the need for the use of multiple medications.
According to our findings, the hospitalization time depends not so much on the severity of the diarrhoea,
but mainly on the decompensation of the general status of the patient. Hospitalization time was frequently
prolonged due to cardiopulmonary insufficiency caused
by the primary protein loss. The presented results point
out the fact that protein deficiency greatly depends on the
duration of the diarrhoea, and not on the number of daily
stools. This should particularly draw the attention of general practitioners, who should consider CDI, confirmed by
rapid and inexpensive tests for the detection of toxins A
and B, in differential diagnosis of moderately-severe cases of diarrhoea (2-3 stools daily) lasting for longer than
14 days. In addition, the diagnostics of protein deficiencies has to be implemented, as they may cause a considerable deterioration in the patient’s general status.
Results from our study indicated that total protein and
albumin levels are an important predictive factor in patients hospitalized due to CDI. It should be pointed out that
an albumin level below 2.5 g/dl is one of the risk factors
for severe cases of CDI (9, 11). Mean total protein level in
the study group was 5.32 g/dl, and 2.85 g/dl for albumins.
In our material, covering a small study group, 28.125% of
patients had albumin levels below 2.5 g/dl. Similar findings (27.7%) were made by researchers who evaluated
a group of 336 patients with CDI (15).
It is worth noting that the priorities of the Polish National Health Plan for 2007-2015 for the prevention of
infectious diseases and infections include, e.g. a reduction in the number of food poisoning cases and
gastrointestinal infections caused by biological factors,
and an improvement in the control of nosocomial infections on a national scale (16). Therefore, the problem
of Clostridium difficile infections requires further investigation, and protein deficiencies in patients with CDI
have to be analysed in detail.
CONCLUSIONS
1. CDI is correlated with protein loss, especially in
the albumin fraction, while the protein deficiency depends on the duration of diarrhoea, not on
the number of stools.
2. CDI is a systemic disease, as suggested by the
increased levels of C-reactive protein.
3. Because of the patient’s age and numerous
concomitant diseases, CDI poses a serious
threat to the patient’s life, and in each case requires the evaluation of protein metabolism.
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received/otrzymano: 10.09.2014
accepted/zaakceptowano: 14.10.2014
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